During the 2009 fiscal year, we accomplished the following: 1. Completed analysis of Emu-deficient germline IgH alleles. These studies demonstrated an epigenetic heirarchy in tissue-specific locus activation;loss of repressive H3K9me2 and gain of activating H3K4me2 mark is Emu-independent, whereas histone acetylation, transcription and transcription-associated epigenetic modifications are Emu-dependent. 2. Demonstrated high density of RNA polymerase flanking Smu sequences in the IgH locus. This was attributed to polymerase pausing and implicated as a mechanism for targeting activation-induced deaminase to the switch region. 3. We utilized chromatin immunoprecipitation to analyze binding of CTCF and the cohesin component Rad27 across the unrearranged IgH locus. We identified two prominent regions of CTCF/cohesin binding between VH and DH gene segments, and in the middle of the VH locus. We are currently testing whether these sites are involved in loop formation that promotes VH recombination. 4. Standardized 3C and 4C assays to study chromosome conformation in pro-B cells. 5. Initiated analysis of DNA methylation state of broken chromosomal intermediates of V(D)J recombination. Developed conditions to analyze DNA methylation of recombination by-products released in the form of circular DNA. 6. Established stromal cell cultures that support B-lymphocyte development in vitro. In this system, we propose to express specific Emu-binding proteins via retroviral gene transfer to identify their role in tissue-specific locus activation. This strategy can also be used to target chromatin remodeling machinery or histone modifying enzymes to IgH BAC transgenes.